Apparatus for transporting biological samples

ABSTRACT

A container assembly for storing, treating, transporting and stabilizing a biological sample includes a container, a cap and a sample holder removably received in the container. The sample holder can be a platform-like device dimensioned to be supported on a ledge formed in the side wall of the container. The sample holder includes a central cavity for receiving the sample and immersing the sample in the stabilizing agent in the container. In another embodiment, the sample holder has a closure member for closing the open top end of the cavity. The container includes a liquid reagent in an amount sufficient to treat the biological sample. The biological sample is retained in a predetermined containment area of the container to maintain the biological sample immersed in the reagent without regard to the orientation of the container.

This application is a continuation of U.S. patent application Ser. No.10/269,094 filed Oct. 11, 2002, which in turn claims priority to U.S.Provisional Application No. 60/328,407, filed Oct. 12, 2001, which arehereby incorporated by reference.

FIELD OF THE INVENTION

The present invention is directed to a method and apparatus forcollecting, transporting, processing and storing biological samples in areagent. The invention is also directed to a method and apparatus fortransporting biological samples where the biological samples arecompletely immersed in a reagent.

BACKGROUND OF THE INVENTION

Biological samples are often obtained by a researcher or clinician fordiagnostic evaluation to determine the presence of certain diseases andto determine an appropriate treatment for the disease. Common diagnosticprocesses for diseases include histological and cytological diagnosis.For example, tumors are typically examined for histological andcytological abnormalities.

Biological samples are also obtained for molecular diagnostic. In recentyears, nucleic acid analysis, and particularly RNA and DNA analysis andstudies have become common place in research for the treatment ofnumerous diseases. An essential requirement for accurate RNA and DNAqualitative and quantitative analysis is the presence of high qualityand intact RNA and DNA. For example, intact nucleic acid is necessaryfor RT-PCR, Northern blot hybridization and nuclease protection assaysanalysis of nucleic acid expressions.

Biological samples can be obtained from various sources and by variousprocesses. Numerous devices exist that are designed to remove a smallamount of tissue from an organ or specimen. For example, small samplescan be obtained using a device similar to a punch to extract corefragments of tissue. Another device for performing a biopsy uses anaspirating needle device that can extract single cells, small cellclumps and tissue fragments.

Generally, it is preferable to perform the histologic or cytologicanalysis immediately after being extracted from the patient or source toobtain the most accurate results possible. Numerous molecular changescan occur in the sample during storage, which can affect the finalresults. For example, nucleic acids in a biological sample can undergonumerous changes, including gene transcription, and the nucleic acidsreadily degrade during storage at room temperature when not treated witha stabilizing agent.

The analysis of a biological sample at the time of collection is oftenimpossible or not practical. Therefore, it is necessary to store thesample under controlled conditions to prevent or inhibit degradation ofthe tissue components and to retain the integrity of the results of theanalysis. Biological samples are typically stored in a container with asuitable fixative reagent. A typical fixative reagent is 10% formaline.Other fixatives include water miscible alcohols, ethanol/acetonemixtures, and ethanol/acetic acid mixtures. Ammonium sulfate solutionshave also been used as disclosed in U.S. Pat. No. 6,204,375 to Lader,which is hereby incorporated by reference in its entirety. Thecontainers with the biological sample in the fixative reagent can thenbe sent to a pathology laboratory or other destination for analysis.

Proper handling of the biological sample is essential for accuratenucleic acid analysis, and particularly for RNA quantitative andqualitative evaluation. The biological samples require an effectiveamount of the fixative reagent to preserve the sample. In addition, somereagents require that the sample be completely covered with the fixativereagent to ensure effective preservation. Typically, the biologicalsamples are simply placed in a small container for storage. Thebiological samples which can be very small can be difficult to locateand recover from the container.

To obtain high quality test results from biological specimens, earlystabilization or preservation of the sample may be required. Biologicalsamples and other cells can be quick frozen by various methods as knownin the art. Specimens for anatomical pathology are typically preservedin formaldehyde and alcohol based solutions. Specimens for moleculartesting have been preserved in these and other reagents, such aschaotropic salts.

Quick freezing of biological samples can be effective in stabilizingcellular and molecular characteristics. Samples are typicallytransported on dry ice. Quick freezing, however, is not always availableor convenient. Typically, the collection location and processinglaboratory are separated in location and time, which creates animpediment to stabilization.

The prior methods and containers for storing, transporting andstabilizing biological samples have experienced some success for theintended purposes, but have several known limitations. There is,however, a continuing need in the industry for an improved container andmethod for storing biological samples.

SUMMARY OF THE INVENTION

The present invention is directed to a method and apparatus forcollecting, transporting, processing and storing biological samples in areagent. The invention is also directed to a method and apparatus fortreating a biological sample with a reagent where the sample iscontinuously immersed in the reagent.

Accordingly, one aspect of the invention is to provide a method formaintaining a biological sample in a stabilizing reagent for stabilizingnucleic acids. The method is particularly suitable for treating wholetissue and cells.

Another aspect of the invention is to provide a method and apparatus fortreating a biological sample with a reagent where the biological samplecan be easily recovered from the reagent.

A further aspect of the invention is to provide a method for collecting,storing, stabilizing, processing and/or transporting a biological samplein a suitable reagent while maintaining a critical ratio of sample andregent.

Another aspect of the invention is to provide a method for stabilizing abiological sample for nucleic acid isolation and molecular diagnosticevaluation.

A further aspect of the invention is to provide a method for storing abiological sample in a nucleic acid stabilizing agent for extendedperiods of time to obtain intact nucleic acid from the sample for theanalysis of RNA expression.

Still another aspect of the invention is to provide a container assemblyfor storing a biological sample, where the container assembly isprefilled with a tissue stabilizing agent.

A further aspect of the invention is to provide a method and containerassembly for harvesting, transporting and storing biological samplesthat is simple and easy to use.

Another aspect of the invention is to provide a container assembly thatis able to accommodate biological samples of different sizes whilecontacting the biological samples with an effective amount of a reagent.

Still another aspect of the invention is to provide a method andapparatus for maintaining a biological sample in a liquid reagent wherethe volume of the sample and the volume of the reagent are maintained ina predetermined ratio sufficient to treat the biological sample.

A further aspect of the invention is to provide a biological samplecontainer assembly having a sample receiving holder of a predeterminedsize to limit and control the size of the biological sample in relationto the amount of a reagent in the container where the reagent isincluded in an amount sufficient to treat the sample effectively.

Still another aspect of the invention is to provide a biological samplecontainer assembly where the assembly reduces the likelihood of spillingthe reagent when the sample is inserted into or removed from theassembly.

Another aspect of the invention is to provide a container assembly fortreating a biological sample in a reagent where the container assemblyincludes a sample holder that fits within a container and is removablefrom the container.

Still another aspect of the invention is to provide a method andapparatus for supporting a biological sample in a liquid reagent withina container where the biological sample remains immersed in the reagentregardless of orientation of the container.

Another aspect of the invention is to provide a kit or packaged assemblyof components for obtaining and treating a biological sample. The kitpreferably includes a sample holder, a container for receiving thesample holder, an amount of a treating reagent, and surgical tools, suchas a scalpel, forceps, and the like. The components of the kit arepreferably clean and sterile and packaged in a suitable sterilepackaging.

A further aspect of the invention is to provide a container assembly forreceiving a biological sample where the assembly includes a containerhaving a closure cap and a removable sample holder having an open topthat is closed by the closure cap when coupled to the container. Thesample holder can be tethered to the container or the closure cap. Inone embodiment, the sample holder can be coupled to the container.

Another aspect of the invention is to provide a biological samplecontainer assembly including a reagent container and a sample holderdimensioned to fit within the container with limited lateral andvertical movement of the sample holder within the container to retainthe sample holder in a predetermined area within the container.

Another aspect of the invention is to provide a biological samplecontainer assembly having a container and a removable sample holderwithin the container, where the sample holder includes a tissuereceiving cavity positioned to retain a biological sample immersed in astabilizing liquid.

A further aspect of the invention is to provide a method and apparatusfor defining a containment area in a volume of a liquid where thecontainment area is oriented to remain immersed in the liquid regardlessof the orientation of the apparatus.

A further aspect of the invention is to provide a biological samplecontainer assembly including a container, a closure member and aremovable sample holder, where the sample holder has at least onesupporting leg and can be supported on an inner face of the closuremember.

A still further aspect of the invention is to provide a biologicalsample container assembly including a container, a closure member and asample holder, where the closure member includes a plunger to immerse abiological sample into the reagent within the container and to displacea predetermined amount of air from the container, thereby reducing theheadspace above the reagent.

Another aspect of the invention is to provide a container assembly for abiological sample including a container and sample holder having acavity enclosed by a permeable surface to enable the free flow of areagent into the cavity around the biological sample. The permeablesurface can be made from a permeable media, such as a mesh material,paper filter or porous membrane screen.

Another aspect of the invention is to provide a biological samplecontainer assembly including a container and a sample holder where thesample holder includes a removable closure.

A further aspect of the invention is to provide a sample holder for abiological sample where the sample holder is dimensioned to fit within acontainer and where the holder includes a permeable portion to enablethe free flow of a reagent into the holder.

Another aspect of the invention is to provide a biological sample holderhaving an open top and a closure pivotally coupled to the holder toclose the vessel and where the holder cooperates with a containercontaining a reagent.

In one embodiment of the invention, the container assembly includes acontainer and sample holder that fits in the container and closuremember. The sample holder is in the form of a platform-like devicehaving an outer peripheral edge that nests on a ledge within a side wallof the container. The sample holder includes a recessed area forsupporting a biological sample. The recessed area is formed by apermeably wall to allow the free flow of the liquid reagent through therecessed area. The closure member includes a body or plunger-like memberthat closes the open top end of the recessed area when coupled to thecontainer. The sample holder includes a plurality of supporting legs forsupporting the sample holder when removed from the container.

In another embodiment, the sample holder is a free standing device thatis removable from the container. The sample holder is formed with apermeably side wall and a permeable bottom forming a cavity with an opentop. A closure cap is coupled to the holder by a hinge for closing thecavity. The cap can include a tab extending upwardly to enable theoperator to remove the sample holder from the container.

These and other aspects of the invention are basically attained byproviding a container assembly for storing a biological sample. Thecontainer has a bottom, a side and an open top end. The container alsohas a dimension to contain a volume of a reagent sufficient to treat abiological sample. A closure member is provided for coupling to the opentop end of the container. A sample holder is removable from thecontainer and closure member and has an internal cavity with a dimensionfor receiving a biological sample. The holder has a plurality of fluidopenings into the cavity to enable free flow of the reagent into thecavity. The sample holder also has a dimension to fit between the bottomand side of the container and the closure and to immerse the cavity inthe reagent.

The aspects of the invention are further attained by providing acontainer assembly comprising a container having a bottom, a side and anopen top end, and being dimensioned to contain a liquid reagent. Aclosure is removably coupled to the container and closes the open topend. The closure has an outer face and an inner face. A sample holderhas an internal cavity for receiving a biological sample. The holder hasat least one fluid opening into the cavity and has a dimension to fitwithin the container to completely immerse the cavity in the liquidreagent and to substantially prevent linear movement of the sampleholder in the container. A body is coupled to the inner face of theclosure to displace a portion of the liquid reagent in the container.

The aspects of the invention are still further attained by providing amethod of stabilizing nucleic acids in cells and biological samplescomprising the steps of providing a container having a bottom, a sideand an open top end. The container contains a nucleic acid stabilizingreagent. A biological sample is placed in a sample holder. The sampleholder has an internal cavity for receiving the biological sample andhas a fluid opening into the cavity. The sample holder is positioned inthe container and is completely immersed the internal cavity in thereagent. A closure member is placed on the container to close thecontainer. The closure member cooperates with the sample holder to limitmovement of the sample holder within the container and to retain theinternal cavity immersed in the reagent.

The various aspects, advantages and other salient features of theinvention will become apparent from the annexed drawings and thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawing, in which:

FIG. 1 is an exploded perspective view of the container assembly in afirst embodiment;

FIG. 2 is an exploded perspective view of the container assembly showingthe sample holder received in the container;

FIG. 3 is an exploded side view of the assembly of FIG. 1;

FIG. 4 is an exploded cross-sectional side view of the containerassembly;

FIG. 5 is a cross-sectional view of the assembled container assembly;

FIG. 6 is a top view of the sample holder;

FIG. 7 is a cross-sectional view of the sample holder of FIG. 6 takenalong line 7-7 of FIG. 6;

FIG. 8 is a top view of the closure member of the container;

FIG. 9 is a bottom view of the container;

FIG. 10 is a cross-sectional side view of the sample holder support onthe closure member;

FIG. 11 is a cross-sectional side view of the container in an uprightposition depicting a containment area below the surface of the reagent;

FIG. 12 is a cross-sectional side view of the container of FIG. 11positioned at an incline;

FIG. 13 is a cross-sectional side view of the container of FIG. 11positioned on its side;

FIG. 14 is a side view of the sample holder in a second embodiment;

FIG. 15 is a cross-sectional view of the sample holder of FIG. 14;

FIG. 16 is a perspective view of the sample holder of FIG. 14 and thecontainer;

FIG. 17 is a cross-sectional side view of the container assemblyincluding the sample holder of FIG. 14;

FIG. 18 is an exploded perspective view of the container assembly inanother embodiment of the invention;

FIG. 19 is a side view of the sample holder of the embodiment of FIG.18;

FIG. 20 is a top view of the sample holder of the embodiment of FIG. 19in the open position;

FIG. 21 is a top view of the closed sample holder;

FIG. 22 is a cross-sectional side view of the sample holder;

FIG. 23 is a side view of the sample holder in the container where thecontainer is shown in cross-section;

FIG. 24 is a side view of the container assembly showing the closuremember supporting the sample holder;

FIG. 25 is a cross-sectional side view of a sample holder in anotherembodiment of the invention;

FIG. 26 is a side view of the container assembly of the embodiment ofFIG. 25;

FIG. 27 is a side view in partial cross section of the containerassembly of FIG. 27 in an upright position;

FIG. 28 is a side view in partial cross section of the containerassembly of FIG. 27 in an inverted position;

FIG. 29 is a front view in partial cross-section of a packaged kitincluding a container, a sample holder and forceps;

FIG. 30 is an exploded side view of the container in an embodiment wherethe sample holder is tethered to the container;

FIG. 31 is an exploded side view of the container in an embodiment wherethe sample holder is tethered to the cap of the container;

FIG. 32 is a side view of the container in a further embodiment of theinvention;

FIG. 33 is a cross-sectional side view of the container assembly of FIG.32;

FIG. 34 is a cross-sectional side view of the container assembly of FIG.32 showing the sample holder coupled to the container;

FIG. 35 is a cross-sectional side view showing the cap coupled to thesample holder in the container;

FIG. 36 is a cross-sectional side view of the container assemblycontaining a biological sample and a liquid reagent; and

FIG. 37 is a cross-sectional view of the container assembly in anotherembodiment showing the sample holder fixed to the container.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method and apparatus forcollecting, transporting, processing and storing biological samples. Theinvention is also directed to a method and apparatus for collecting,transporting, processing and storing biological samples in a liquidreagent where the samples are completely immersed in the reagent.

The method and apparatus of the invention are particularly suitable fortransporting biological samples, such as biological samples intended formolecular diagnostic processes. The biological samples are retained in acontainer assembly that contains a suitable reagent, such as, forexample, a liquid stabilizing agent, so that the biological sampleremains completely immersed in the reagent regardless of the orientationof the container. In other embodiments, the reagent can be a gel, solidor semi-solid which can be in the form of beads or other particles. Thesolid of gel can be used alone or in combination with a liquid reagent.

The invention is particularly directed to a method of treating abiological sample with a suitable reagent in a container. The methodensures that the sample contacts an amount of the reagent effective totreat the sample and to maintain the sample immersed in the reagent toprevent or minimize contact of the sample with air. In one embodiment, acontainer is filled to a predetermined level with a liquid reagent sothat when the sample is placed in the container, the sample displaces aportion of the reagent and raises the level of the reagent to a levelsufficient to completely immerse the sample without spilling thereagent. Preferably, the sample is retained in a predetermined locationor area in the internal cavity of the container so that the sampleremains immersed in the reagent regardless of the orientation of thecontainer.

In one embodiment, the method of treating a biological sample with areagent includes the steps of collecting a biological sample andimmediately placing the sample in the reagent. Typically the sample iscompletely immersed in the reagent without any intermediate steps.Preferably the sample is completely covered by or immersed in thereagent as soon as possible after collection to minimize contact withthe air. The sample is retained in a retaining area in a container thatcontains the reagent so that the sample remains immersed during handlingand transporting of the container without the sample being exposed tothe air.

Referring to FIGS. 1-10, the invention, in a first embodiment, isdirected to a container assembly 10. Container assembly 10 includes acontainer 12, a closure cap 14 and a sample receiving holder 16.

In the embodiment illustrated, container 12 has a substantiallycylindrical shape formed by a side wall 18 and a bottom wall 20. Sidewall 18 extends from an open top end 22 of container 12 to a bottom edge24. A plurality of recessed areas 26 are formed in side wall 18 adjacentbottom edge 24. Recesses 26 have a dimension to assist the operator ingripping the container for assisting the operator in opening and closingcontainer assembly 10.

Side wall 18 includes external threads 28 adjacent open top end 22 formating with complementing threads on cap 14. A rib 30 extends radiallyoutward from side wall 18 and is spaced axially from open top end 22.Rib 30 preferably is spaced from open top end 22 a distancecomplementing the dimensions of closure cap 14 and encircles container12.

Referring to FIG. 4, container 12 has an internal cavity 32 with adimension sufficient to contain an effective amount of a liquidstabilizing agent sufficient to stabilize a biological sample. In theembodiment illustrated, bottom wall 20 is spaced from bottom edge 24 ofside wall 18 by a cylindrical portion 33 to form a recess 34 in thebottom of container 12. In alternative embodiments, the bottom ofcontainer 12 can be substantially flat.

Side wall 12 includes a ledge 36 extending in a substantially radialdirection with respect to a center axis of container 12. Ledge 36 has awidth sufficient to support sample holder 16 as discussed hereinafter ingreater detail. In the embodiment illustrated, ledge 36 is positioned atthe upper end of side wall 18 and is oriented substantially parallel toa top edge 38 of container 12. Preferably, ledge 36 is formed in sidewall 18. In alternative embodiments, a rib can extend inwardly from theinner face of side wall 18 a distance to support sample holder 16.

Cap 14 has a dimension complementing side wall 18 of container 12 formating with container 12. As shown in FIGS. 3 and 4, cap 14 includes aside wall 40 having a substantially cylindrical shape with an outersurface 42 and an inner surface 44. As shown in FIG. 3, outer surface 42includes a plurality of dimples 46 to assist the operator in handlingcap 14. Inner surface 44 includes threads 48 for mating with threads 28of container 12.

Cap 14 includes a top wall 50 coupled to side wall 40. An annular rib 52extends in a generally downward, axial direction with respect to cap 14from top wall 50. Rib 52 includes an inner axial side wall 54 and anouter axial side wall 56 connected by bottom wall 55. As discussedhereinafter, bottom wall 55 of rib 52 is oriented to contact sampleholder 16 while in container 12 to limit movement of sample holder 16.Outer axial side wall 56 extends substantially parallel to side wall 40and is spaced inwardly from side wall 40 a distance to form a recess 58for mating with top edge 38 of container 12.

Top wall 50 of cap 14 includes a centrally located body defining aplunger 60 having a substantially frustoconical shaped bottom wall 62and an annular side wall 64 extending between top wall 50 and bottomwall 62. As discussed hereinafter, plunger 60 defines a closure memberfor sample holder 16. As shown in FIG. 4, side wall 64 of plunger 60 hasan axial length slightly greater than the axial length of side wall 40and rib 52 so that bottom wall 62 of plunger 60 is spaced axiallyoutward from the bottom edge of side wall 40. Side wall 64 of plunger 60is spaced from inner axial side 54 of rib 52 to form an annular shapedrecess 66. In one preferred embodiment, side wall 64 of plunger 60 has aslightly tapered frustoconical shape.

An annular lip 68 extends upward from top wall 50 adjacent the outeredge in an axial direction as shown in FIG. 4. Lip 68 forms a ledge withtop wall 50 and is dimensioned to complement bottom edge 24 of side wall18 of container 12. In this manner, several container assemblies 10 canbe stacked vertically with the bottom edge 24 of wall 18 received in thearea defined by lip 68.

Sample holder 16 is dimensioned to be received in container 12 and tosupport a biological sample. Sample holder 16 in the embodiment of FIGS.1-10 includes a top wall 70 having a circular outer edge 72 and an inneredge 74. Inner edge 74 defines a central opening 76 into a cavity 78 forreceiving a biological sample. Outer edge 72 of top wall 70 has adimension complementing the inner dimension of container 12. An annularridge 80 extends axially upward from a top face 81 of top wall 70adjacent inner edge 74 and encircles central opening 76. A plurality ofopenings 82 are formed in top wall 70 adjacent annular ridge 80 as shownin FIGS. 1 and 2. A pair of tabs 84 are coupled to top wall 70 andextend in a substantially axial direction with respect to top wall 70from top face 81. Tabs 84 have a dimension and are oriented to enablethe operator to lift and manipulate sample holder 16.

Cavity 78 of sample holder 16 is formed by a body 86 having an openbottom end 88 and a plurality of side openings 90. Open bottom end 88include a permeable material, such as a screen or porous mesh 92, todefine a bottom end of cavity 78. Side openings 90 also include apermeable or porous mesh 94 to form porous sides of body 86. Preferably,porous mesh 92 and 94 are made from a nylon mesh having a pore size toretain a biological sample in cavity 78 and to allow liquid to passthrough. Other permeable materials, such as filter paper, can be used toenclose cavity 78 and retain the biological sample.

A plurality of legs 96 extend downwardly from a bottom face 83 of topwall 70 and are integrally formed with body 86. In the embodimentillustrated, legs 96 have a generally planar configuration and areoriented in a plane extending radially outward from a center axis ofsample holder 16. Legs 96 are spaced apart a distance to support andstabilize sample holder 16 when placed on a horizontal surface.

Preferably, legs 96 have an axial length greater than an axial length ofbody 86 and extend beyond body 86 in an axial direction as shown inFIGS. 3 and 4. Legs 96 have a top end 98 coupled to top wall 70 and abottom end 100 with a notch 102 formed along the bottom and the outeredge of legs 96.

Container assembly 10 is preferably made of a suitable plastic materialthat is non-reactive with the stabilizing agents and does not interferewith the biological sample. The components of container assembly 10 aregenerally made by a suitable injection molding process as known in theart.

In one embodiment of the invention, container assembly 10 is prefilledwith a liquid reagent 106. Container assembly 10 is sealed, packaged andshipped to the physician or clinician for receiving and transporting abiological sample. Container assembly 10 can include a suitable seal ortamper indicator. In other embodiments, container assembly 10 can bepackaged without a reagent and shipped to the consumer empty. Cap 14 isremoved and container 12 is filled with a suitable reagent at the timeof use. In one embodiment, container 12 is prefilled with the reagent106 and sample holder 16 is packaged separately in a suitable sterilepackage. The sample holder is removed from the sterile package and thebiological sample is placed in the holder. The container with thereagent is removed from its sterile package and the sample holder withthe biological sample is placed in the reagent.

In use, the operator removes cap 14 to expose sample holder 16. Cap 14can be inverted and placed on a horizontal surface 106 as shown in FIG.10. Sample holder 16 is then removed from container 12 by gripping tabs84 and lifting upwardly to remove sample holder 16 from the reagent.Bottom end 100 of legs 96 of sample holder 16 are oriented to nest inrecess 66 of cap 14 as shown in FIG. 10. In this manner, the liquidreagent adhering to sample holder 14 will drain into the recesses of cap14. In addition, bottom end 100 of legs 96 engage recess 66 of cap 14 tostabilize sample holder 16 during use.

A biological sample 108 is then placed in cavity 78 of sample holder 16.In alternative embodiments, sample holder 16 can remain in container 12and biological sample 108 deposited directly into cavity 78 and intoreagent 106. Typically, it is desirable to separate sample holder 16from container 12 at the time the biological sample is placed in sampleholder 16 to prevent splashing of the reagent.

As shown in FIG. 5, outer edge 72 of top wall 70 of sample holder 16 issupported by ledge 36 of container 12. In the illustrated embodiment,container 12 and sample holder 16 are dimensioned so that bottom end 100of legs 96 are spaced from bottom wall 20 of container 12. Outer edge 72of top wall 70 has a dimension complementing the inner dimension of sidewall 18 to limit movement of sample holder 16 within container 12.

In alternative embodiments, legs 96 can rest on bottom wall 20 ofcontainer 12 to support the sample holder 16 within the container 12. Inthis embodiment the side wall of the container can be formed without theledge. The outer dimension of top wall 70 of sample holder 16 has adimension to fit in the container and limit lateral movement between theside of the container.

With sample holder 16 positioned in container 12, cap 14 is mated withcontainer 12 to close open top end 22. As shown in FIG. 5, plunger 60 ofcap 14 has a dimension complementing opening 76 in top wall 70 of sampleholder 16. Plunger 60 effectively closes cavity 78 to retain biologicalsample 108 in cavity 78 when cap 14 is coupled to container 12. Bottomwall 55 of rib 52 engages sample holder 16 as shown in FIG. 5 to limitaxial movement of sample holder 16 within container 12.

As shown in FIG. 5, plunger 60 is oriented to extend inwardly intocontainer 12 in an axial direction below top end 22 of side wall 18. Ascap 14 is threaded onto container 12, plunger 60 displaces a volume ofair and a volume of the reagent and reduces the head space above reagent106 while closing central opening 76 of top wall 70. Plunger 60 ispreferably dimensioned to push biological sample 108 downwardly intoreagent 106 to retain biological sample 108 immersed in reagent 106 andto raise the level of reagent 106 within container 12. Preferablyplunger 60 has a dimension to form a retaining area in cavity 78 in alocation so that the sample remains immersed in the reagent.

Container assembly 10 is particularly suitable for containing a liquidreagent for preserving and stabilizing a biological sample. To ensureadequate contact of biological sample 108 with reagent 106, body 86 ofsample holder 16 preferably includes permeable mesh 92 and 94 to allowcontinuous circulation of reagent 106 through cavity 78. The volume ofreagent 106 necessary to effectively treat biological sample 108 candepend on the nature and concentration of the stabilizing agent. Inpreferred embodiments, the ratio of the volume of container 12 to thevolume of cavity 78 is at least 5:1, and typically ranges from about 5:1to about 12:1. Preferably, the ratio of the volume of stabilizing agent104 in container 12 to the volume of cavity 78 containing biologicalsample 108 is about 10:1. This ensures a suitable ratio of at least 10:1of the volume of the reagent and the biological sample.

Cavity 78 of sample holder 16 has a dimension to contain an appropriatesize and dimension of a biological sample. In one embodiment, cavity 78is about 2 cm in diameter and about 0.75 cm deep. Preferably, cavity 78has a dimension to receive a sample having at least one dimensionranging from 1 mm to about 5 cm. For example, cavity 78 can have adimension to receive a core needle biological sample having a length ofabout 3 cm and a diameter of about 1-2 mm. Cavity 78 can also bedimensioned to receive larger samples ranging from about 3.5 cm to about5 mm in at least one dimension.

Biological sample 108 can be removed from sample holder 16 by removingcap 14 to expose cavity 78. Typically, cap 14 is placed on a horizontalsurface 106 as shown in FIG. 10 and sample holder 16 is removed fromcontainer 12 and placed on cap 14. Permeable mesh 92 and 94 preferablyhave a sufficiently small pore size to enable holder 16 to strain smallpieces of the biological sample. Top wall 70 of sample holder 16 has agenerally frustoconical shape and is inclined in a generally downwarddirection toward central opening 76. Reagent 106 is directed along topwall 70 toward drain openings 82. Reagent 106 is collected in therecesses of cap 14 and is either discarded or poured back into container12. Biological sample 108 then can be readily removed from cavity 78 andanalyzed according to standard analytical processes as known in the art.

The method of the invention contacts a biological sample with a treatingreagent in a minimum predetermined ratio to ensure contact of the tissuesample with an effective amount of the reagent sufficient to treat thesample. The volume of the tissue sample is controlled in relation to theamount of the reagent to regulate the relative amount of the biologicalsample to the amount of the reagent. In preferred embodiments of theinvention, the ratio of the relative volume of the reagent to thebiological sample is at least 5:1. Typically, the ratio of the volume ofreagent to the volume of the biological sample is at least about 10:1.The critical amount of the reagent to treat a biological sampleeffectively can vary depending on the particular sample and theparticular reagent. The amount of reagent required to treat a sampleeffectively is affected by the weight, volume and density of the sample.For example, some tissue samples are dense compared to other tissues andmay require more or less of a particular reagent than that required by aless dense or porous tissue.

The biological samples that are treated by the methods of the inventionare typically tissue samples. Examples of biological samples that can betreated include organ specimens, tumor specimens, bone specimens, andconnective tissue specimens, such as tendons and membranes.

The reagent for treating the biological sample is preferably a liquidbut can be a gel or viscous material. The treating reagent is typicallyan aqueous or alcohol solution containing a suitable reagent, such as astabilizing agent or fixative reagent. Examples of suitable reagentsincluding stabilizing agents, lysing agents, drying agents, preservationreagents, and cationic detergents. The reagents can be organic orinorganic compounds. In one embodiment, the reagent is a 10% by volumeaqueous formaline solution.

The method and apparatus of the invention are particularly suitable foruse in transporting a biological sample to another location, such as toa remote laboratory, while stabilizing and preserving the sample. Themethod of the invention in one embodiment collects a biological sample,such as a tissue sample, and immediately places the sample in thecontainer assembly to immerse the sample in the reagent contained withinthe container assembly. The container assembly is able to retain thebiological sample immersed in the reagent while being transported and toprovide an amount of the reagent sufficient to treat the sample. In oneembodiment, the reagent is a nucleic acid stabilizing reagent that isable to preserve the nucleic acids in the cells of the sample forextended periods of time. Preferably, the sample is collected andimmediately immersed in the stabilizing reagent to enable high qualityquantitative and qualitative analysis of the nucleic acids.

The reagent is preferably an aqueous medium or alcohol containing one ormore components for treating the biological sample. In one embodiment,the reagent is for stabilizing cells and biological samples. In onepreferred embodiment, the preserving and stabilizing reagent is able topreserve nucleic acids for extended periods of time prior to isolationfrom the cells. The stabilizing reagent included in the container is anamount effective to penetrate the cells and biological sample to preventor inhibit nucleases from decomposing the nucleic acids.

In one embodiment, the reagent is able to precipitate nucleic acids andthe cellular protein in the sample to inhibit or inactivate the actionof the nuclease. In this embodiment, the stabilizing agent is an aqueousmedium containing a salt that is able to precipitate the nucleic acidand cellular proteins. Examples of suitable salts are sulfates, such asammonium sulfate, ammonium bisulfate, cesium sulfate, cadmium sulfate,cesium iron (II) sulfate, chromium (III) sulfate, cobalt (II) sulfate,copper (II) sulfate, lithium sulfate, magnesium sulfate, manganesesulfate, potassium sulfate, sodium sulfate and zinc sulfate. The saltconcentration can range from about 0.10 to 1.50 g/ml, and preferablyabout 0.7 g/ml. In other embodiments, the stabilizing agent can includeformalin or a chaotropic salt such as quanidium compounds.

The reagent can also include amounts of ethanol, methanol, acetone,trichloracetic acid, propanol, polyethylene glycol, acetic acid and achelating agent such as EDTA. Buffering agents such as sodium acetatecan also be added. Generally, the stabilizing agent has a pH of about4-8.

The reagents are generally liquids that can pass through the porous meshof the sample holder to contact the biological sample. In otherembodiments, the reagent can be a gel, solid or semi-solid in the formof beads or particles. The gel and the beads can be permeable orimpermeable to the porous mesh of the sample holder. Typically the beadsor particles have a particle size that is larger than the opening sizeof the mesh in the sample holder and are not permeable though the mesh.In embodiments where the gel or beads are impermeable to the mesh of thesample holder, the cavity of the sample holder can contain an amount ofthe gel, beads or particles to contact the biological sample. Thedimensions of the cavity are selected to provide the necessary volumeratio of the biological sample to the reagent. The gel can be permeableto the permeable mesh of the sample holder to pass through the walls ofthe sample to contact and immerse the biological sample in the reagent.The solid or semi-solid reagent, such as beads or particles, can be usedalone although they are typically used in combination with a liquid orgel reagent to supplement the solid or semi-solid reagent so thebiological sample is maintained immersed in the reagents. In thisembodiment, the cavity of the sample holder can contain the solid orsemi-solid reagent and the container can contain the liquid or gelreagent that is permeable to the wall of the sample container. In thismanner the biological sample is placed in the sample holder in contactwith the solid or semi-solid reagent. The sample holder is then placedin the container with the liquid or gel reagent to enable the reagent toflow through the walls of the sample holder to fill the spaces betweenthe beads or particles and to surround the biological sample.

Container assembly 10 is constructed to define an internal containmentarea within container 12 to contain biological sample 108 in apredetermined area of container 12. Preferably, a containment area 109is defined by cavity 78 of sample holder 16. Referring to FIGS. 11-13,sample containment area 109 is shown schematically by phantom lines incontainer 12. As shown in FIG. 11, containment area 109 is oriented insubstantially the center of container 12 between the sides of container12, cap 14, and bottom wall 20. As shown, reagent 106 is filled to alevel to completely immerse containment area 109. Containment area 109and reagent 108 are selected and controlled to maintain containment area109 completely immersed in reagent 109 without regard to the orientationof container 12 as shown in FIGS. 12 and 13. In this manner, thebiological sample will remain completely immersed in the reagent duringhandling and transporting of the container. Containing the biologicalsample in an area that is consistently below the level of the treatingreagent regardless of the orientation of the container substantiallyprevents the biological sample from contacting the air in the container.Retaining the biological sample completely immersed in the treatingreagent enhances complete treatment of the sample with the reagent andminimizes inaccuracies in the test results that can occur when thesample is exposed to air even for short periods of time. Certainreagents, such as nucleic acid stabilizing reagents, are most effectivewhen the sample is immediately immersed in the reagent. Exposing thebiological sample to air can lower the accuracy of the nucleic acidanalysis.

Embodiment of FIGS. 14-17

FIGS. 14-17 illustrate a second embodiment of a container assembly 110in accordance with the invention. Container assembly 110 includes acontainer 112, a closure cap 114 and a sample holder 116. Container 112and cap 114 are substantially the same as container 12 and cap 14 of theembodiment of FIGS. 1-10 so that identical components are identified bythe same reference number with the addition of a prime. In thisembodiment, container 112 has a slight frustoconical shaped bottom walland a substantially straight side wall.

Sample holder 116 includes a top wall 118 having a substantiallyfrustoconical shape converging toward a central opening 120 as shown inFIGS. 14 and 15. An annular wall 122 extends from top wall 118 aroundcentral opening 20 in a substantially upward axial direction. A liquidpermeable mesh 124 is coupled to a bottom end 126 of annular wall 122 todefine a cavity 128 for receiving a biological sample. An annular ridge130 extends upward from central opening 120 in an axial direction. Aplurality of drain openings 132 are formed in top wall 118 adjacentridge 130. A pair of tabs 134 extend upwardly from the top face of topwall 118 for lifting and manipulating sample holder 116.

Legs 136 extend from the bottom face of top wall 118 downwardly in agenerally axial direction. As shown in FIG. 17, legs 136 are spacedradially outward from annular wall 122 and have an axial length greaterthan the axial length of annular wall 122. Legs 136 in the embodimentillustrated have a substantially arcuate shape extending around a centeraxis and are oriented on opposite sides of sample holder 116 forsupporting sample holder 116 in an upright position.

Sample holder 116 is dimensioned to fit within container 112 so thatouter edge 138 of top wall 118 is nested in ledge 36′ of container 112as shown in FIG. 17. Preferably, legs 136 of sample holder 116 have alength so that the bottom ends 140 are spaced from bottom wall 20′ ofcontainer 112. Cap 114 is coupled to container 112 so that plunger 60′closes cavity 128 and displaces air in container 112 to raise the levelof the stabilizing agent above cavity 128 as shown in FIG. 17. Plunger60′ preferably has an axial length to push the biological sampledownward into cavity 128 to retain the biological sample submerged inthe reagent.

In the embodiments of FIGS. 1-10 and 14-17, a sample holder having acavity for receiving a biological sample fits within a container forcontaining a liquid stabilizing agent. The sample holder is retained inthe container by the closure cap. The closure cap closes the open topend of the cavity of the sample holder and closes the container in amanner to retain the biological sample completely immersed in thereagent. In preferred embodiments, the cap displaces a sufficient amountof air from the head space and displaces a portion of the reagent in thecontainer to raise the level of the reagent above the cavity of thesample holder. In addition, displacing air in the head space above thereagent ensures that the biological sample is completely submerged inthe reagent. Preferably, the container contains a sufficient amount ofthe reagent so that the biological sample remains immersed regardless ofthe orientation of the container assembly. The sample holder is pressedagainst the ledge in the side wall of the container to limit axial andlateral movement of the sample holder within the container. The cavityof the sample holder defines a containment area within the container toretain the biological sample below the surface of the reagent.

Embodiment of FIGS. 18-24

Another embodiment of the invention is shown in FIGS. 18-24. Referringto FIG. 18, container assembly 150 includes a container 152, a closurecap 154 and a sample holder 156.

Container 152 has a cylindrical side wall 158 having an upper end 160defining an open top end 162. A bottom wall 164 is coupled to a bottomend 166 of side wall 158. Side wall 158 and bottom wall 164 define aninternal cavity 168 for receiving sample container 156. Upper end 160 ofside wall 158 includes threads 170 on the outer face for mating with cap154.

Cap 154 has a top wall 72 and a depending side wall 174. Side wall 174includes threads 176 on the inner face as shown in FIG. 23. Side wall174 has a dimension to mate with upper end 160 of container 152.

Sample holder 156 is dimensioned to fit in container 152 and is easilyremoved from cavity 168. Preferably, sample holder 156 has a width tonest in container 152 to limit lateral and axial movement of sampleholder 156 within container 152 when cap 154 is coupled to container152. Referring to FIG. 23, sample holder 156 is retained withincontainer 152 to limit movement of sample container 156 while containedwithin container 152.

Sample holder 156 has a body 178 having an internal cavity 180 and anopen top end 182. Body 178 includes a side wall 184 extending from opentop end 182 to a base 186. As shown in FIG. 19, base 186 is flared in agenerally outward direction from side wall 184 to a substantially flatbottom.

Referring to FIG. 22, an internal ledge 188 extends radially inward froman inner surface of side wall 184. A permeable material such as a porousmesh 190 is coupled to ledge 188 to define a liquid permeable bottom ofcavity 180. Ledge 188 and mesh 190 separate cavity 180 from a hollowportion 192 of base 186 and space cavity 180 from the bottom ofcontainer 152 when sample holder 156 is positioned in container 152.

Side wall 184 includes a plurality of spaced-apart openings 194 coveredby a porous mesh 196 to enable a reagent to flow through cavity 180.Base 186 also includes a plurality of spaced-apart openings 198 to allowthe flow of a liquid reagent into hollow portion 192 and through mesh190 into cavity 180.

A plurality of tabs 200 extend radially outward from side wall 184 ofbody 178. In the embodiment illustrated, four tabs 200 are uniformlyspaced around side wall 184. Tabs 200 have a generally flat planarconfiguration and are oriented in a plane extending in an axialdirection and in a radial direction with respect to a center axis ofbody 178. As shown in FIG. 23, tabs 200 have an outer edge 202 defininga width of sample holder 156 and define the position of sample holder156 within container 152. Tabs 200 function as stabilizing members toposition sample holder 156 in container 152.

Side wall 184 of body 178 includes a radially extending flange 204spaced from open top end 182 to define an annular collar 206. Sampleholder 156 includes a closure member such as a cap 208 for closing opentop end 182 of cavity 80. In a preferred embodiment, cap 208 is coupledto body 178 by flexible hinge 210. Preferably, cap 208 is integrallyformed with body 178 by a suitable plastic molding process. Hinge 210 iscoupled to cap 208 by a tab 212 and is coupled to flange 204 by a tab214.

Cap 208 includes an annular side wall 216 having a bottom end 218 withan outwardly extending radial flange 220. Side wall 216 has an innerdimension complementing the outer dimension of collar 206. In theembodiment illustrated, side wall 216 includes a detent 222 on an innersurface for providing an interference fit of cap 208 with flange 204.

Side wall 216 includes a top end 224 with an inwardly extending radialflange 226. Flange 226 forms an opening 228 which is covered by apermeable mesh 230. A tab 232 forming a handle extends upwardly fromflange 226 in an axial direction with respect to sample holder 156. Tab232 includes a top end 234 defining a height of sample holder 156.Preferably, the height of sample holder 156 complements the height ofcontainer 152 so that when cap 154 is coupled to container 152, sampleholder 156 is permitted limited axial movement within container 152 sothat cavity 180 is retained within a predetermined area in container152.

In one embodiment, container 152 is prefilled with a liquid reagent atthe time of assembly and packaging of container assembly 150. When readyto be used by the technician or scientist, cap 154 is removed fromcontainer 152 and placed on a horizontal surface as shown in FIG. 24.Sample holder 156 is removed from container 152 and placed in theinverted cap 154. In this manner, the stabilizing agent in cavity 180can drain into cap 154. Cap 208 of sample holder 156 is opened bypivoting about hinge 210. A biological sample can then be placed incavity 180. Cap 208 is pivoted to the closed position and snapped ontocollar 206. Sample holder 156 is then replaced in container 152 so thatcavity 180 is immersed in the liquid reagent 136 as shown in FIG. 23.

Sample holder 156 is dimensioned to contain a biological sample ofsufficient size for analysis by standard procedures. Preferably, cavity180 has a dimension sufficient to receive the biological sample and issufficiently open to the reagent to allow the stabilizing agent to flowthrough the cavity to ensure complete immersion of the biological samplein the reagent. Typically, the ratio of the volume of container 152 tothe volume of cavity 180 is about 10:1. Tab 232 of cap 208 preferablyhas a dimension to be easily gripped by the technician using forceps orother tools for lifting sample holder 156 from container 152. Anaperture 238 can be provided to assist in gripping tab 232 formanipulating sample holder 156.

Embodiment of FIGS. 25-28

FIGS. 25-28 show another embodiment of the container assembly 250 forreceiving a biological sample. Container assembly 250 includes acontainer 252, a closure cap 254 for the container 252 and a sampleholder 256.

Container 252 is similar to the container of the previous embodimentsand includes cylindrical side wall 258 having an open top 260 withexternal threads 262 for mating with closure cap 254. Container 252 hasa bottom wall and is dimensioned to receive sample holder 256 and tocontain an effective amount of a treating liquid. Cap 254 has a top wall266 and a side wall 268 with internal threads 270 for mating withthreads 262 of container 252.

Sample holder 256 in this embodiment has a density greater than thedensity of the treating reagent 272 so that sample holder 256 will sinkin the reagent and remain in the bottom of the container in allorientations. As in the previous embodiments, sample holder 256 has adimension to fit within container 252 as shown in FIGS. 27 and 28 and incap 254 as shown in FIG. 26.

Referring to FIGS. 25 and 26, sample holder 256 has a body 274 formed bya side wall 276 defining an internal cavity 278. Body 274 has an opentop end 280 that is closed by a removable closure member 282.

Body 274 of sample holder 256 includes a base 284 having an axialpassage 186 and a slightly rounded bottom end 288. Base 284 defines aweighted portion to ensure that sample holder 256 sinks in the reagent272. In the embodiment illustrated, side wall 276 includes an internalledge 290 for supporting a permeable screen or mesh 292. Side wall 276also includes a plurality of openings 294 that are closed by a permeablescreen 296 to enclose internal cavity 278. A plurality of openings 296are provided in body 274 to allow the flow of the reagent to axialpassage 286.

A plurality of tabs 298 extend outwardly from side wall 276 of body 274.Tabs 298 have a planar configuration and are oriented in a substantiallyaxial direction of body 274. Preferably, tabs 298 have a dimension tocomplement the inner dimension of container 252 to limit lateralmovement of sample holder 256 within container 252.

Side wall 276 of body 274 includes a collar 300 for coupling withclosure member 256. Closure member 282 has an annular wall 302 with aninner dimension complementing collar 300 for coupling closure member 282to collar 300. Closure member 282 can be coupled to collar 300 by afriction fit or an interference fit. Preferably, closure member 282 isconnected to body 274 by a flexible hinge 304 that is integrally moldedwith body 274 and closure member 282. Alternatively, a two-part hingewith a hinge pin can be used.

Closure member 282 is formed with an opening 306 in the top end andincludes a liquid permeable mesh 308 to close opening 306. A handle 310is coupled to closure member 282 to assist in lifting and manipulatingsample holder 256.

Cavity 278 of sample holder 256 is dimensioned to contain a biologicalsample. Cavity 278 has an internal volume to limit the size of thebiological sample so that the size of the biological sample iscontrolled in relation to the volume of the reagent in container 252.Preferably, ratio of the volume of the reagent in container 252 to thevolume of the biological sample is at least 5:1, and preferably at least10:1.

Container assembly 250 is used in a similar manner as in the previousembodiments. Sample holder 256 can be placed in cap 254 as shown in FIG.26 so that the reagent can be collected. The closure member 282 isopened and a biological sample is placed in cavity 278 of sample holder256. Sample holder 256 is closed and placed in container 252 containingreagent 272. Container 252 contains an amount of liquid reagent 277 tocover sample holder 256 in any orientation of container 252 as shown inFIGS. 27 and 28. Sample holder 256 has a density sufficient to sink tothe bottom of container 252 regardless of the density of the biologicalsample contained in sample holder 256 as shown in FIGS. 27 and 28.

Embodiment of FIG. 29

FIG. 29 shows an embodiment of the invention in the form of aprepackaged sterile kit 310 for use by the physician or clinician. Thekit 310 includes a package 312 enclosing a container 314, a sampleholder 316 and one or more tools, such as a pair of forceps 318.Preferably, the components are clean and sterile to be ready for use.Package 312 in the illustrated embodiment is a plastic pouch formed asheet of plastic film or material that can be heat sealed around one ormore of the edges 320. The heat sealed edges 320 can be formed as peellayers that can be readily separated by the operator to remove thecontents.

In one embodiment, container 314 is prefilled with a liquid reagent andsample holder 316 packaged separately from container 314 as shown inFIG. 29. Alternatively, sample holder 316 is packaged in container 314and removed from container 314 prior to use. In the embodimentillustrated, a pair of forceps is included in package 312, althoughother tools and surgical instruments can be included such as a scalpeland measuring gauge.

Embodiment of FIGS. 30 and 31

FIGS. 30 and 31 illustrate another embodiment of the invention. Acontainer assembly 330 includes a container 332, a closure cap 334 and asample holder 336.

Container 332 is similar to the containers of the previous embodimentsand include a cylindrical side wall 338 having an upper end 340 definingan open top 342 and a bottom wall 344. Upper end 340 includes threads346 for coupling closure cap 334 to container 332. Cap 334 has a topwall 348 and a depending side wall 350. Side wall 350 includes internalthreads to mate with threads 346 on container 332.

Sample holder 336 is dimensioned to fit in container 332 to hold abiological sample in the reagent contained in container 332. In oneembodiment, sample holder 336 has a width and height to limit lateraland longitudinal movement in container 332.

Sample holder 336 has a body 352 having an internal cavity and an opentop end 354. Body 352 includes a side wall 356 extending from the opentop end to a base 358. As shown in FIGS. 30 and 31, base 358 is flaredin a generally outward direction from side wall 356 to a substantiallyflat bottom.

As in the previous embodiments, the bottom wall of the sample holder 336has a permeable material such as a porous mesh 360 to define a liquidpermeable bottom.

Side wall 356 includes a plurality of spaced-apart openings 362 coveredby a permeable material such as a porous mesh 364 to enable a reagent toflow through the cavity. Base 358 also includes a plurality ofspaced-apart openings 366 to allow the flow of a liquid reagent throughthe mesh into the cavity.

Side wall 356 of body 352 includes a radially extending flange 368spaced from open top end 354 to define an annular collar 370. Sampleholder 336 includes a closure member such as a cap 372 for closing opentop end 354 of the cavity. In a preferred embodiment, cap 372 is coupledto body 352 by flexible hinge 374. Preferably, cap 372 is integrallyformed with body 352 by a suitable plastic molding process.

Cap 372 includes an annular side wall 376 having a bottom end with anoutwardly extending radial flange. Side wall 376 has an inner dimensioncomplementing the outer dimension of collar 370. A post 371 extendsdownwardly from cap 372 a distance sufficient to position the biologicalsample in the cavity of sample holder 336 to keep the biological samplesubmerged in the reagent.

A tab 378 forming a handle extends upwardly from the top surface of cap372 in an axial direction with respect to sample holder 336. Tab 378includes a top end defining a height of sample holder 330. Preferably,the height of sample holder 336 complements the height of the containerso that when cap 372 is coupled to the container, sample holder 336 ispermitted limited axial movement within the container so that the cavityis retained within a predetermined area in the container. In oneembodiment, sample holder 336 can be removably coupled to a bottom wallof container 332 to maintain sample holder 336 in a fixed location whiletransporting the biological sample.

In the embodiment of FIGS. 30 and 31, sample holder 336 is tethered tocontainer 332 as in FIG. 30 or to cap 334 as in FIG. 31. A string 380 orstrip of flexible material has a first end connected to cap 334 orcontainer 332 and a second end extending through a hole 382 in tab 378.In this embodiment, sample holder 336 is connected to a component ofassembly 330 that supports a label or other identifying indicia foridentifying a biological sample. In this manner, the sample holder iscontinuously tethered to an identifying marker on the cap or container.In another embodiment, the container or lid is provided with a writablesurface so that the identification can be written or printed on thecontainer or cap.

Embodiments of FIGS. 32-37

Another embodiment of the invention is shown in FIGS. 32-37. Referringto FIG. 32, container assembly 386 includes a container 388, a closurecap 390 and a sample holder 392. In the embodiment of FIGS. 32-36,sample holder 392 is removably coupled to container 388 so that sampleholder 392 remains in a fixed position within container 388. Sampleholder 392 of FIGS. 32-36 is separable from container 388 for filingwith a biological sample, after which sample holder 392 is returned andcoupled to container 388. In the embodiment of FIG. 37, sample holder392 is permanently fixed to container 388. Typically, sample holder 392is integrally molded with container 388 in the embodiment of FIG. 37.

Container 388 has a cylindrical side wall 394 having an upper end 396defining an open top end 398. A bottom wall 400 is coupled to a bottomend of side wall 394. Side wall 394 and bottom wall 400 define aninternal cavity 402 for receiving sample holder 392. Upper end 396 ofside wall 394 includes threads 402 on the outer face for mating with cap390.

Cap 390 has a top wall 404 and a depending side wall 406. Side wall 406includes threads on the inner face as shown in FIG. 33. Side wall 406has a dimension to mate with upper end 396 of container 388.

Sample holder 392 is dimensioned to fit in container 398 and is easilyremoved from cavity 402. Sample holder 392 has a body 408 having aninternal cavity 410 and an open top end 412. Body 408 includes a sidewall 414 extending from open top end 412 to a base 416. As shown in FIG.33, base 414 has a substantially cylindrical configuration with anoutwardly extending radial rib 418. Bottom wall 400 of container 388includes an annular recess 420 dimensioned to receive base 416 of sampleholder 392 in a coupling relationship. In the embodiment illustrated,recess 420 is defined by concentric walls 422. The outermost wall 422has an inwardly open groove 424 to receive rib 418 of base 414 tosecurely couple sample holder 392 to container 388. Sample holder 392 isremovably coupled to container 398 by a press fit, such as aninterference fit or friction fit.

In one preferred embodiment, sample holder 392 is securely attached tocontainer 388 by a snap-fit between rib 418 and groove 424 to fix thelocation of sample holder 392 within container 388. Recess 420 formed bywalls 422 define a coupling member for removably coupling sample holder392 to container 388. Sample holder 392 is separable from container 388by pulling upward with sufficient force to overcome the resistancebetween rib 418 and groove 424. In the embodiment shown in FIG. 37,sample holder 392 is integrally formed with container 388 and is notseparable. Container 388 includes a writable surface for identificationof the biological sample. Sample holder 392 can be separated fromcontainer 388 by pulling upward on sample holder 392 with respect tocontainer 388.

Referring to FIGS. 33 and 34, an internal ledge 426 extends radiallyinward from an inner surface of side wall 414. A permeable material suchas a porous mesh 428 is coupled to ledge 426 to define a liquidpermeable bottom of cavity 410. Ledge 426 and mesh 428 separate cavity410 from a hollow portion 430 of base 416 and space cavity 402 from thebottom of container 388 when sample holder 392 is coupled to container388 as shown in FIGS. 33 and 34.

Side wall 414 includes a plurality of spaced-apart openings 432 coveredby a porous mesh 434 to enable a reagent to flow through cavity 402.Base 416 also includes a plurality of spaced-apart openings 436 to allowthe flow of a liquid reagent into hollow portion 430 and through mesh428 into cavity 402.

Side wall 414 of body 408 includes a radially extending flange 438spaced from the open top end to define an annular collar 440. Sampleholder 392 includes a closure member 442 in the form of a cap forclosing the open top end of cavity 410. In the illustrated embodiment,cap 442 is separable from body 408 and is removably coupled to body 408by a snap-fit.

Cap 442 includes an annular side wall 444 having a bottom end with anoutwardly extending radial flange 446. Side wall 444 has an innerdimension complementing the outer dimension of collar 440. In theembodiment illustrated, side wall 444 includes a rib 448 on an innersurface for providing an interference fit of cap 442 with collar 440 ofbody 408.

Referring to FIG. 33, side wall 444 of cap 442 includes a top end withan inwardly extending radial flange 450. Flange 450 forms openings 452which are covered by a permeable mesh 454. A tab 456 forming a handleextends upwardly from flange 450 in an axial direction with respect tosample holder 392. Tab 456 includes a top end defining a height ofsample holder 392. Preferably, the height of sample holder 392complements the height of container 388 so that when cap 442 is coupledto container 388, sample holder 392 is spaced closely to cap 390.

In one embodiment, container 388 is prefilled with a liquid reagent atthe time of assembly and packaging of container assembly. When ready tobe used by the technician or scientist, cap 390 is removed fromcontainer 388 and cap 442 is removed from body 408 of sample holder 392to expose cavity 410. In one embodiment, container 388 is filled to alevel to fill cavity 410 completely as shown in FIG. 34. Typically, thereagent level has a depth to immerse the biological sample in thereagent without the sample floating out of cavity 410. The biologicalsample is placed in cavity 410 and cap 390 is snapped onto body 408 ofholder 392.

The biological sample can be placed in sample holder 392 while sampleholder 392 remains coupled to container 388. Cap 442 of sample holder392 can be removed from sample holder 392 without removing sample holder392 from container 388. In this embodiment, the cavity of sample holder392 is filled with the reagent. The biological sample is collected andplaced directly into the reagent in the cavity of the sample holder 392to minimize contamination and exposure of the biological sample to air.In alternative embodiments, sample holder 392 is separated from recess420 and removed from container 388. Sample holder 392 can be placed inthe inverted cap 390 to collect the reagent. The biological sample canbe placed in the sample holder 392. Cap 442 is placed on sample holder392. Sample holder 392 is then returned to the container 388 to immersethe biological sample in the reagent. Preferably, sample holder 392 issecured in the recess in container 388 to maintain sample holder 392 ina fixed location during shipping and handling of the assembly.

In the embodiment illustrated, cap 442 includes a post 458 extendingdownwardly from tab 456 into cavity 410 of sample holder 392 as shown inFIG. 34. Typically, post 458 is oriented to extend along the axis ofcavity 410 and generally in the axial center of cavity 410. Post 458 hasa substantially cylindrical shape with an axial end 460. Axial end 460includes teeth 462 to engage the biological sample 464 and maintain thesample 464 immersed in the liquid reagent 466 in container 388. In oneembodiment, post 458 has a width and length to capture biological sample464 between axial end 460 of post 458 and bottom mesh 428 to fix thelocation of biological sample 464 during storage.

While various embodiments have been chosen to demonstrate the invention,it will be understood by those skilled in the art that variousmodifications and additions can be made without departing from the scopeof the invention as defined in the appended claims.

1. A container assembly for storing a cell or biological sample, saidassembly comprising: a container having a side, an open top end, and aninternal dimension to contain a volume of a reagent sufficient to treata biological sample; a cap for coupling to and closing said open top endof said container; and a sample holder situated within said containerhaving a closure member and an internal cavity with a dimension forreceiving a biological sample, said sample holder having a plurality offluid openings into said cavity to enable free flow of said reagent intosaid cavity, said sample holder having a dimension to fit in saidcontainer below said cap and to immerse said cavity in said reagent,wherein said sample holder is detached from said cap and said containerduring use of said container assembly, wherein said closure membercooperates with said cap to ensure said internal cavity of said sampleholder remains immersed in said reagent.
 2. The container assembly ofclaim 1, wherein said sample holder includes a permeable materialdefining said fluid openings.
 3. The container assembly of claim 1,wherein said sample holder has a density greater that a density of saidreagent to sink said sample holder in said container.
 4. The containerassembly of claim 1, wherein said cap includes a member to displace aportion of said reagent in said container.
 5. The container assembly ofclaim 1, wherein said closure member has an inner surface and a postextending from said inner surface and being oriented to extend into saidinternal cavity of said sample holder.
 6. The container assembly ofclaim 5, wherein said post is oriented to extend in an axial directioninto said internal cavity toward a bottom end of said sample holder. 7.The container assembly of claim 5, wherein said post extends into saidinternal cavity of said sample holder to capture a biological samplebetween said post and a bottom end of said sample holder.
 8. Thecontainer assembly of claim 5, wherein said post has an axial end and atleast one projection from said axial end to capture a biological sample.9. The container assembly of claim 1, wherein said closure member ispivotally coupled to said sample holder to pivot between an open andclosed position.
 10. The container assembly of claim 1, wherein saidclosure member includes a permeable material to enable the flow of saidreagent into said sample holder.
 11. The container assembly of claim 10,wherein said side and said bottom of said sample holder include apermeable material to enable said reagent to flow into said sampleholder.
 12. The container assembly of claim 1, wherein said sampleholder includes a base in contact with a bottom of said container andspacing said internal cavity of said sample holder from said bottom ofsaid container.
 13. The container assembly of claim 1, wherein saidclosure member of said sample holder includes a top face and a handlemember extending from said top face.
 14. The container assembly of claim1, wherein said sample holder includes at least one stabilizing memberextending outward from a side of said sample holder, said at least onestabilizing member having a dimension such that upon contact of said atleast one stabilizing member with said container said internal cavity ofsaid sample holder remains immersed in said reagent.
 15. The containerassembly of claim 14, comprising a plurality of said stabilizing membersextending in a substantially radial outward direction from said side ofsaid sample holder.
 16. A container assembly for storing a cell orbiological sample, said assembly comprising: a container having a side,an open top end, and an internal dimension to contain a volume of areagent sufficient to treat a biological sample; a cap for coupling toand closing said open top end of said container; and a sample holdersituated within said container, having a closure member and an internalcavity with a dimension for receiving a biological sample, said sampleholder having a plurality of fluid openings into said cavity to enablefree flow of said reagent into said cavity, said sample holder having adimension to fit in said container below said cap and to immerse saidcavity in said reagent, said sample holder being detached from said cap,wherein during use of said container assembly, said sample holderremains unconnected to said cap, wherein said cap contacts said sampleholder to position said internal cavity of said sample holder immersedin said reagent, wherein said sample holder includes at least onestabilizing member extending outward from a side of said sample holder,said at least one stabilizing member having a dimension such that uponcontact of said stabilizing members with said container said internalcavity of said sample holder remains immersed in said reagent.
 17. Thecontainer assembly of claim 16, comprising a plurality of saidstabilizing members extending in a substantially radial outwarddirection from said side of said sample holder.